Several imaging methods are known for diagnostic purposes, including the use of radioisotopes or x-ray imaging. Besides these methods, diagnostic imaging is performed on biopsies of tissues or liquids from the living body to determine the existence or cause of a disease. In the last decade imaging systems were developed using cell cultures of cells taken from a patient, in order to detect markers that are correlated to diseases. These systems are mostly based on the coupling of biological samples to defined epitopes wherein the biological samples are labelled with a dye, preferably a fluorescent dye.
Pharmacological testing and low throughput screening assays are also increasingly based on mammalian cell culture (High content screening, HCS). As compared to biochemical affinity tests used in molecular high-throughput screening (HTS), these HCS-bioassays offer the smallest living unit for detecting adverse effects on a least damage level. Thus, they can help to avoid stressing animals with pharmaceutical lead compounds. Usually, also in cell based assays, the readout is a biochemical one like DNA-, RNA- and protein-chips or immunocytochemistry or sometimes electrophysiological experiments.
Atomic force microscopy (AFM) was invented two decades ago and became a versatile tool for biological studies on single biomolecules, aggregates, viruses, cells or tissues. The method bridges the gap between the nm-resolution technique electron microscopy (EM) and the μm-scale optical microscopy (OM). As such, it combines the advantages of high resolution and the ability to investigate cells under physiological buffer conditions. At the same time, no disadvantageous sample drying or coating as necessary for EM or chromophore labelling as in fluorescence microscopy (FM) are needed. Additionally, local mechanical properties of the sample can be obtained (Riethmuller C., Schaffer T. E., Kienberger F., Stracke W. and Oberleithner H.; 2007; Ultramicroscopy 107:895-901; Rotsch C. and Radmacher M.; 2000; Biophys. J. 78:520-535). Hence, investigation of biological specimen very close to physiological conditions is possible with only a minimum of procedure-derived artifacts.
Recording the topography of biological specimen including cellular surfaces—either living or fixed—has been the basis of biologically inspired AFM studies ever since (Braet F., de Zanger R. and Wisse E.; 1997; J. Microsc. 186 (Pt 1):84-87.; Oberleithner H., Giebisch G. and Geibel, J.; 1993; Pflugers Arch. 425:506-510; Chang L., et al.; 1993; Biophys J. 64: 1282-1286). But distinct structures are difficult to identify, especially on whole cells, where only cytoskeletal structures are obviously recognisable. They almost exclusively consist of fibrillary actin, contributions of microtubuli can be neglected. However, since morphological features are difficult to classify, a reproducible quantification is hard to perform. Therefore, most studies use a qualitative, rather descriptive approach., so that often an overlay of AFM and fluorescence marker is desired to prove the claimed structure.
However, the fluorescence identification is hampered by mutually disturbing fixation protocols for AFM and FM. Unequivocally accepted structural characteristics are actin stress fibers (Haga H., Sasaki S., Kawabata K., Ito E., Ushiki T. and Sambongi T.; 2000; Ultramicroscopy 82:253-258), microvilli (Poole K., Meder D., Simons K. and Muller D.; 2004; FEBS Lett. 565:53-58) and cell junctions (Riethmuller C., Oberleithner H., Wilhelmi M., Franz J., Schlatter E., Klokkers J. and Edemir B.; 2008; Biophys. J. 94:671-678). Recently, also intracellular organelles could be identified through their specific mechanics (Riethmuller C., Schaffer T. E., Kienberger F., Stracke W. and Oberleithner H.; 2007; Ultramicroscopy 107:895-901). Beyond that, only very delicate setups can deliver more detailed information; in special cell models, where one type of receptor is abundantly expressed, some aggregates can be imaged marker-free (Hoogenboom, B. W., Suda K., Engel A. and Fotiadis D.; 2007; J. Mol. Biol. 370:246-255) or with the TREC procedure, where topography can be recorded simultaneously with localization of one specific target using antibody-modified tips (Kienberger F., Ebner A., Gruber H. J. and Hinterdorfer P.; 2006; Acc. Chem. Res. 39:29-36).